Starch extracted from corn, cassava, rice, potatoes and wheat serves as a major source of raw materials for the large-scale production of sugars and derivatives in the starch industry. General starch is usually made up of two types of macromolecules, amylose and amylopectin, and the relative amounts of each mainly depend on the source species. Amylose is a linear polysaccharide comprised of glucose residues linked only by α-1,4-glucosidic bonds, whereas in amylopectin, besides the α-1,4-glucosidic bonds, glucose residues are also joined by α-1,6-glucosidic linkages to form branch points. In order to degrade the starch and obtain simple sugars, the starch is initially depolymerized by heat stable α-amylase, which partially hydrolyzes the α-1,4-glucosidic bonds, followed by a saccharification step, in which the smaller branched and linear units are further converted into glucose or maltose by addition of a glucoamylase or beta-amylase (Norman, 1982).
It has been proposed that the addition of a debranching enzyme that is capable of hydrolyzing α-1,6-glucosidic bonds during the saccharification step of starch can yield higher purity glucose and maltose syrups. Meanwhile, the debranching enzyme can reduce saccharification time and increase the applied substrate concentrations (Bakshi et al., 1992). Nowadays, this application has been widely used in industry, such as for starch conversion, beer brewing, and amylose production.
Pullulanase (pullulan 6-glucanohydrolases, EC 3.2.1.41) is classified as a debranching enzyme that specifically hydrolyzes α-1,6-glucosidic bonds in starch, pullulan, and related branched polysaccharides. Given the growing demand for the improvement of such enzymatic technology and reduction of production costs during the saccharification of starch, seeking improved pullulanases that are more efficient in starch conversion has become an important area for both industry and academia.
Many microbial pullulanases have been found and characterized from plants and bacteria, including Klebsiella pneumonia (d'Enfert, Ryter et al. 1987), Fervidobacterium pennavorans (Koch. Canganella et al. 1997), Thermoactinomyces thalpophilus (Odibo et al. 1988), and Bacillus species (Nakamura, Watanabe et al. 1975). Modified pullulanase enzymes derived from bacterial pullulanases have also been reported (e.g., U.S. Pat. No. 7,906,306, U.S. Pat. No. 7,449,320, and U.S. Pat. No. 7,968,691). For example, U.S. Pat. No. 7,449,320 reports a mixture of truncated forms of pullulanases derived from a native bacterial pullulanase (SEQ ID NO: 25) having N-terminal deletions of 98 and 102 amino acid residues obtained from cleavage of the mature pullulanase by extracellular proteases of the recombinant host cell. This mixture was reported to be most stable at a pH of 4.5. However, the truncated forms were not isolated, nor was the activity of the mixture compared to the activity of the untruncated mature form.
U.S. Pat. No. 7,968,691 discloses a truncated pullulanase derived from a native bacterial pullulanase having an N-terminal deletion of 104 amino acids. Pullulanase activity was tested by transforming a plasmid encoding the truncated pullulanase into B. subtilis, and screening for halo formation in a pullulan overly assay (0.1% in 100 mM NaAc pH 5.0, 1%).
The most commercially valuable pullulanases are pullulanases from Bacillus species, particularly Bacillus acidopullulyticus (Lappalainen et al., 1991; Kusano et al., 1988) and Bacillus deramificans (Deweer et al. U.S. Pat. No. 6,074,854, 2000). These pullulanases have a molecular mass of about 100 kD, which is similar to pullulanases obtained from other sources, and have the ability to hydrolyze α-1,6-glucosidic bonds at an acidic pH at 60° C. Although suitable for the production of high-purity glucose and maltose in the starch industry, the pullulanases from Bacillus acidopullulyticus and Bacillus deramificans exhibit a slow saccharification rate, and decreased enzyme activity at increased temperatures and low pH, particularly at temperatures over 60° C. and pH values lower than 4.5, conditions that are often used for controlling industrial processes.
Accordingly, there exists a need in the art for improved pullulanase enzymes that have an increased saccharification rate, and improved enzymatic activity at temperatures over 60° C. and acidic pH values below 4.5.